Hemoglobin (Hb) plays a central role in biological oxidation by efficiently transporting O2, the vital oxidant, from the lung to the tissues and CO2, one of the major waste products of metabolism, from the tissues to the lung. Thus, understanding the molecular mechanism of how Hb regulates its functions is of biomedical significance. The two-state allosteric model of Monod, Wyman, and Changeux (MWC) and Perutz has been long considered the most plausible description of a wide variety of structural and functional data on the cooperative O2 binding to Hb under physiological conditions. Recent work performed under the auspices of this grant has challenged the fundamental assumption of the MWC/Perutz model, namely, that the O2 affinity of Hb is primarily controlled by the T/R quaternary structural transition. Work performed in our laboratory shows that a new "global allostery" model is required to fully account for the large variations of oxygenation properties observed in the presence of heterotropic allosteric effectors. This "global allostery" model proposes that the tertiary structural changes induced by the interactions of Hb in both T (deoxy) and R (oxy) states with heterotropic allosteric effectors primarily modulate functions of Hb such as the O2 affinity, cooperativity, and Bohr effect. This proposal aims at investigating the molecular functions of Hb based upon the "global allostery" model using thermodynamic, kinetic, calorimetric, spectroscopic, structural, and computational techniques in order to establish a viable molecular mechanism for cooperativity and allostery in Hb that explains the functional behavior of Hb from a global viewpoint. Elucidation of the molecular mechanism of allostery of Hb will undoubtedly contribute to our further understanding of the mechanisms of allosteric enzymes which play vital roles in the control and regulation of metabolic processes.